Inhibition of HERG K+ current and prolongation of the guinea-pig ventricular action potential by 4-aminopyridine

Abstract

4-Aminopyridine (4-AP) has been used extensively to study transient outward K+ current (ITO,1) in cardiac cells and tissues. We report here inhibition by 4-AP of HERG (the human ether-à-go-go-related gene) K+ channels expressed in a mammalian cell line, at concentrations relevant to those used to study ITO,1. Under voltage clamp, whole cell HERG current (IHERG) tails following commands to +30 mV were blocked with an IC50 of 4.4 +/- 0.5 mM. Development of block was contingent upon HERG channel gating, with a preference for activated over inactivated channels. Treatment with 5 mM 4-AP inhibited peak IHERG during an applied action potential clamp waveform by ~59 %. It also significantly prolonged action potentials and inhibited resurgent IK tails from guinea-pig isolated ventricular myocytes, which lack an ITO,1. We conclude that by blocking the alpha-subunit of the IKr channel, millimolar concentrations of 4-AP can modulate ventricular repolarisation independently of any action on ITO,1.

Figure 1. Concentration and voltage-dependent inhibition of I_HERG by 4-AP

A, upper traces show representative current traces illustrating the effect of different concentrations of 4-AP. Lower trace represents voltage protocol used. B, concentration response relation for I_HERG tail blockade by 4-AP (n = 5–8 cells at each concentration). C, representative records in the absence (Ci) and presence (Cii) of 5 mm 4-AP. D, the mean (± s.e.m.) fractional block of I_HERG by 5 mm 4-AP over a range of potentials (n = 8 cells). I_HERG activation curves are shown in the absence (continuous line) and presence (dashed line) of 5 mm 4-AP.

Figure 2. Time dependence of I_HERG block by 4-AP

A, traces from an ‘envelope of tails’ protocol, in the absence (Ai) and presence (Aii) of 5 mm 4-AP. Lower traces represent the voltage protocol used. B, the mean (± s.e.m.) fractional block of I_HERG with varying pulse duration (n = 5 cells). The τ for development of inhibition was 32.3 ± 6.4 ms (n = 5).

Figure 3. Effect of strong depolarisation on the action of 4-AP

A, current records (upper traces) elicited by the voltage protocol shown (lower trace), applied from a holding potential of −80 mV. Arrows indicate traces obtained in control and 4-AP. B, bar charts (n = 5 cells) compare the mean level of blockade at 1000 ms into protocol (at +20 mV), at 4000 ms (at +80 mV) and at 9000 ms (after membrane potential was returned to +20 mV).

Figure 4. Effect of 4-AP under action potential conditions

A, representative records of I_HERG, after P/4 leak subtraction, (upper traces) activated by ventricular action potential command (lower trace), in control and in the presence of 5 mm 4-AP. B, effect of 5 mm 4-AP on action potentials from guinea-pig ventricular myocytes. C, effect of 4-AP on resurgent I_K tails from a guinea-pig ventricular myocyte (upper traces; lower trace shows a section of the voltage protocol. A mean inhibition of 71.1 ± 4.3 % was observed (n = 6).